Three-dimensional constructs - Stromal Cells: HCFs and HKCs were plated at a density of 1 × 106 cells/well on six-well size polycarbonate membrane inserts with 0.4-μm pores (VWR, Radnor, PA, USA). The cells were cultured in EMEM containing 10% FBS, 1% antibiotic, and stimulated with a stable Vitamin C derivative (0.5 mM 2-O-α-d -glucopyranosyl-l -ascorbic acid: Sigma-Aldrich, St. Louis, MO, USA). Cultures were grown for a total of 4 weeks and fresh media was supplied every other day for the duration of the study. Protein extraction and further analysis occurred at the 4-week timepoint.
Two-dimensional conventional cultures - Epithelial Cells: Cells were brought up from liquid nitrogen and cultured in a T75 flask in epithelial media, consisting of Keratinocyte Growth Media 2 PromoCell (VWR, Radnor, PA, USA), Penicillin-Streptomycin Solution (ThermoFisher, Rockford, IL, USA), and CaCl2 (Sigma-Aldrich, St. Louis, MO, USA). Once confluent, cells were removed via trypsinization and seeded in a 6-well plate at 1 × 106 cells/well in epithelial media. Cells were cultured at 37 °C for 24 h before protein was extracted [27 (link),28 (link)].
Two-dimensional conventional cultures - Epithelial Cells: Cells were brought up from liquid nitrogen and cultured in a T75 flask in epithelial media, consisting of Keratinocyte Growth Media 2 PromoCell (VWR, Radnor, PA, USA), Penicillin-Streptomycin Solution (ThermoFisher, Rockford, IL, USA), and CaCl2 (Sigma-Aldrich, St. Louis, MO, USA). Once confluent, cells were removed via trypsinization and seeded in a 6-well plate at 1 × 106 cells/well in epithelial media. Cells were cultured at 37 °C for 24 h before protein was extracted [27 (link),28 (link)].
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